title: Identification of a vitamin D3-specific hydroxylase genes through actinomycetes genome mining.
abstract: We previously completed whole-genome sequencing of a rare actinomycete named Sebekia benihana, and identified the complete S. benihana cytochrome P450 complement (CYPome), including 21 cytochrome P450 hydroxylase (CYP), seven ferredoxin (FD), and four ferredoxin reductase (FDR) genes. Through targeted CYPome disruption, a total of 32 S. benihana CYPome mutants were obtained. Subsequently, a novel cyclosporine A region-specific hydroxylase was successfully determined to be encoded by a CYP-sb21 gene by screening the S. benihana CYPome mutants. Here, we report that S. benihana is also able to mediate vitamin D3 (VD3) hydroxylation. Among the 32 S. benihana CYPome mutants tested, only a single S. benihana CYP mutant, ΔCYP-sb3a, failed to show regio-specific hydroxylation of VD3 to 25-hydroxyvitamin D3 and 1α,25-dihydroxyvitamin D3. Moreover, the VD3 hydroxylation activity in the ΔCYP-sb3a mutant was restored by CYP-sb3a gene complementation. Since all S. benihana FD and FDR disruption mutants maintained VD3 hydroxylation activity, we conclude that CYP-sb3a, a member of the bacterial CYP107 family, is the only essential component of the in vivo regio-specific VD3 hydroxylation process in S. benihana. Expression of the CYP-sb3a gene exhibited VD3 hydroxylation in the VD3 non-hydroxylating Streptomyces coelicolor, implying that the regio-specific hydroxylation of VD3 is carried out by a specific P450 hydroxylase in S. benihana.
authors.affiliation: Department of Biological Engineering, Inha University, Incheon, 402-751, Korea.
authors.forename: Jun-Gyu
authors.initials: JG
authors.lastname: Ban
authors.forename: Hyun-Bum
authors.initials: HB
authors.lastname: Kim
authors.forename: Mi-Jin
authors.initials: MJ
authors.lastname: Lee
authors.forename: Periasamy
authors.initials: P
authors.lastname: Anbu
authors.forename: Eung-Soo
authors.initials: ES
authors.lastname: Kim
mesh_terms: D000192:Actinomycetales / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D002762:Cholecalciferol / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D016680:Genome, Bacterial; D006900:Hydroxylation; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D048372:Streptomyces coelicolor / Q000235:genetics / Q000378:metabolism
chemical_list: D002762:Cholecalciferol; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases